Solid phase support synthesis of oligomers such as polynucleotides, polypeptides, and polysaccharides is currently the synthetic method of choice. Solid phase support synthesis allows for efficient removal of reactants from a growing oligomer by simple washing excess reagent from the reaction product that is attached to the solid phase support. Additionally, synthesis on a solid phase support allows for semi-automation of the synthetic process.
Various solid supports have been studied for oligonucleotide synthesis. Early efforts were directed to organic resins, especially polystyrene resins. A review of these early efforts is found in Amarnath, V. and Broom, A. D., Chemical Reviews, 1977, 77:183-219. Later-developed styrene polymer supports include graft polymers disclosed in U.S. Pat. No. 4,908,405 to E. Bayer and W. Rapp. The graft polymers of Bayer and Rapp include graft polymers of polyoxyethylene and polyvinyl alcohol, polyhydroxy-styrene or chloromethylated polystyrene crosslinked with ethylene glycol, oligoethylene glycol, polyacrylate polymer, polymethacrylate polymer functionalized with hydroxy groups. These are noted as being of importance for peptide synthesis. The polyoxyethylene chains of such graft polymers are high molecular weight chains having molecular weights from 500 to 50,000.
In U.S. Pat. No. 4,373,071, K. Itakura describes the use of a polyacrylmorpholide support resin for oligonucleotide synthesis. The use of a polystyrene resin is also disclosed. Itakura compares the polystyrene resin to the polyacrylmorpholide resin and notes that the polystyrene resin is advantageous because it is not hydrophilic, i.e. "it has no problems of affinity to hydroxylic groups such as water, methanol and ethanol." In this patent and in U.S. Pat. No. 4,401,796 Itakura further describes a derivatized polystyrene resin. This derivatized resin is formed by treatment of chloromethyl-polystyrene with potassium phthalimide to form the corresponding phthalimido methyl resin. The phthalimido methyl styrene resin is then converted to an amino methyl styrene resin by treatment with hydrazine in ethanol. A 2'-deoxynucleotide is attached to the amino methyl styrene resin by activation of the nucleotide via treatment with succinic anhydride. The monosuccinate derivatized nucleotide is then treated with pentachlorophenol and dicyclohexylcarbodimide in DMF to form an activated ester. The activated ester reacts with the amino methyl styrene resin to link the nucleotide to the amino functionality of the resin. The nucleotide-resin link is achieved by way of an succinyl amide linkage. Further use of an amino methyl styrene resin is described in U.S. Pat. No. 4,507,433 to P. S. Miller and P.O.P. Ts'O.
A process of functionalizing polystyrene to its amino methyl derivative is also described by Lieto, J., Milstein, D., Albright, R. L., Minkiewicz, J. V. and Gates, B. C., Chemtech, 1983, 13(1):46. The authors derivatize polystyrene via chloromethylation followed by amination of the chloromethyl compound with an amine. A primary amino group is introduced via derivatization with gaseous, anhydrous ammonia. Derivatization with secondary and tertiary amino moieties is also described.
In a first publication in 1973 (Seliger, H. and Aumann, G., Tetrahedron Letts., 1973, 31:2911-2914) and in two subsequent publications in 1975 (Seliger, H. and Aumann, G., Die Makromolekulare Chemie, 1975 176:609-627 and Seliger, H., Die Makromolekulare Chemie, 1975 176:1611-1627), a copolymer of N-vinylpyrrolidone and vinylacetate is described. Post polymerization, the vinylacetate is hydrolyzed to vinyl alcohol. The vinyl alcohol-N-vinylpyrrolidone copolymer was then used for synthesis of thymidyl homopolymers. A thymidine nucleotide was attached to the hydroxyl group of the copolymer via reaction of a 5' chloroformate derivatized nucleotide. This results in nucleotide attachment via a carbonate linkage. The authors reported synthesis of a dimeric, trimeric, tetrameric and a pentameric homopolymer of thymidine. The reported overall yields of these short homopolymers were low; the pentamer was reported in 6% yield. No mixed oligonucleotides nor polymers larger than the pentamer were reported.
Inorganic supports have also been utilized for oligonucleotide solid-phase synthesis. These employ silicon base compounds as the structural support element. U.S. Pat. No. 4,812,512 suggests a derivatized form of Kieselguhr, i.e. silica gel, as well as derivatized forms of polytetrafluoroethylene, cellulose and metallic oxides. The Kieselguhr is appropriately treated to attach a linker thereon. The linker includes alkyl chains linked by ether, amide and/or sulfonamide moieties. The linker also includes a terminal amino group that is used as the attachment point for attaching a nucleotide to the support such as by utilizing succinic anhydride. The succinyl moiety connects between the nucleotide and the linker forming an ester linkage with the 3' hydroxyl group of the nucleotide and an amide linkage with terminal amine of the linker. Other silica based supports include Fractosil. These supports, however, can cause blockage of sintered glass funnels utilized during the synthetic cycle of an oligonucleotide synthesis. Furthermore, the non-uniform pore size of these supports can result in failure products.
Presently the most commonly utilized solid phase support material for use in oligonucleotide synthesis are the commercially available CPG (controlled pore glass) supports available from CPG Incorporated. Such controlled pore glass consists of uniformly milled and screened particles of almost pure silica that are hydrated under acidic conditions to produce particles with uniform pore size. Beads of a selected size are derivatized with a long chain having a terminal alkyl amine functionality. As described above for the silica gel support, succinic anhydride is used to attach a first nucleotide of the desired oligonucleotide sequence to the support.
CPG supports allow for the practice of synthetic chemistry based upon the published protocols of M. H. Cruthers and his associates. This chemistry utilizes tetrazole activated phosphoramidites as the reactive species for the synthesis. The procedure has been standardized to an extent that allows non-chemistry trained personal to operated commercial "synthesizers" such as those available from Applied Biosystems Inc., Foster City, Calif. Generally an ester group is utilized to attach the 3' hydroxyl group of the 3' terminal nucleotide of the oligonucleotide to the solid phase support. As described above this ester is generated from one of the two carbonyl groups of a succinyl moiety. The other carbonyl group of the succinyl moiety in turn is connected to the solid phase support via groups that are more stable than the ester such that when the oligonucleotide is removed from the solid phase support, cleavage of the oligonucleotide from the support occurs cleanly at the ester yielding a 3'-OH group on the 3' terminal nucleotide of the oligonucleotide.
CPG supports, however, are not without their problems. Loading is typically in the range of 30-50 .mu.mol of oligonucleotide per gram of support due to limited surface area. While increasing the pore size increases surface area, the resulting support is brittle, crushing easily and clogging frits during synthesis. A polystyrene/polyoxyethylene graft polymer as described above was shown to have a loading capacity of 150-200 .mu.mol/gram. Other Merrifield resins can have up to 1000 .mu.mol/gram loading. It is evident that the CPG supports exhibit limited loading when compared to other support systems. Because of this limited loading, for large scale synthesis on CPG support, cost become a major concern since the price of this support is typically in the range of about $50,000.00 per kilogram of support.
Because it contains silicon atoms, CPG is highly hydrated and is hydrophilic. Solid-phase phosphite oligonucleotide synthesis utilizes phosphoramidites as the active species for phosphitylation. These phosphoramidites are easily hydrolyzed and thus deactivated. The oxidization step during an oligonucleotide cycle introduces water. Additionally atmospheric moisture can be absorbed in solvents or vacuumed through the reaction vessels. After the oxidization step, capping is practiced. Excess capping reagent (an anhydride) is used to theoretically scavenge any residual water from the oxidization step. Even though extreme care is taken to attempt to completely remove all water after the oxidization, the kinetic barrier to removal of all the water from the CPG support is difficult to overcome and water is retained on the CPG support. This thus requires that a large (up to 20 fold) excess of the phosphoramidites be used for each nucleotide added to an oligonucleotide. Since the phosphoramidate reagents are also very expensive, this further serves to increase the cost of effecting large scale oligonucleotide synthesis on CPG support.
Other problems are also encountered with CPG supports. Certain of these are reviewed in European Patent application 0 375 278. This patent suggests using non-swellable porous polystyrene supports to circumvent the problems associated with CPG supports. Such non-swellable porous polystyrene achieves it non-swellable characteristics by virtue of being an insoluble polymer.
Kinetic considerations favor solution phase reactions over solid phase reactions since with a solid phase reaction, a highly ordered layer of solvent becomes bound to the surface of the solid phase that inhibits free diffusion to target molecules bound to the solid support. This bound phase essentially encapsulates target molecules on the surface of the solid phase supports. Reagents must traverse through this bound phase to react with target molecules attached to the surface of the solid phase. In solution phase reactions, the target molecules are generally less encapsulated with bound solvent.
Soluble polymers behave much like solution phase reactions. Compared to insoluble polymers they do not exhibit as great a layer of bound solvent as do insoluble polymers. However, non-swelling polymers have reduced loading capacity. Furthermore, depending upon the composition of the polymer, soluble polymers however can exhibit different degrees of swelling in different solvents. Since many synthetic reaction schemes, including those utilized for solid phase oligonucleotide synthesis, require the use of various solvents throughout the synthetic scheme, unequal swelling of the polymer support during different parts of the synthetic cycle can result in variability between synthetic cycles or loss of yield.
Thus, a polymer which optimizes swelling, giving increased loading, while limiting solvent layering, and equalizing swelling between different solvents is greatly desired.